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Pitt Team Reports on Viral Use of Host microRNA to Evade Immune Detection

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New research out of the University of Pittsburgh has uncovered how a virus uses of a vertebrate microRNA to suppress its own replication in certain cells in order to avoid detection by its host's immune system.

The findings, which were published last month in Nature, offer insights into why the virus — eastern equine encephalitis virus (EEEV) — is so deadly and may give clues how to treat the infection, according to the study's authors.

North American strains of EEEV are highly virulent, mosquito-borne alphaviruses, and wild-type EEEV is known to be defective for replication in human and mouse macrophages and dendritic cells, the Pitt team wrote. It is characterized by limited early symptoms prior to encephalitis, resulting from restricted myeloid cell replication and minimal induction of systemic type I interferon.

In cell culture experiments, the investigators used a luciferase-expressing translation reporter RNA encoding the 5' and 3' non-translated regions and translation initiation control sequences of the wild-type virus to show that translation was restricted in a mouse monocyte/macrophage myeloid cell line known as RAW 264.7, though not in BHK-21 fibroblasts. Translation of an analogous reporter RNA derived from the related Venezuelan equine encephalitis virus (VEEV), however, was efficient in both types of cells.

Notably, removal of the EEEV 5' NTR did not impact the restriction of translation in the RAW cells, suggesting that it was the 3' NTR that conferred this characteristic. In line with this reasoning, they discovered that transfer of the EEEV 3' NTR to a host mRNA mimic triggered the translation blockade in RAW cells but, again, not in the fibroblasts.

Using miRNA prediction algorithms, the scientists identified four potential binding sites for a hematopoietic cell-specific miRNA, known as miR-142-3p, in the 3' NTR of one North American EEEV strain. Importantly, three canonical seed sites of the miRNA are conserved in 17 out of the 23 sequences EEEV strains collected since 1954, indicating "a strong selection for their retention," they wrote in Nature.

To confirm if it is these binding sites in the EEEV 3' NTR that restrict the virus' replication, the group generated an EEEV mutant with a deletion of 260 nucleotides encompassing all of the miR-142-3p binding sites. In the BHK-21 cells, there was no significant difference in viral replication. In the RAW cells, however, replication was nearly 1,000-fold higher than in wild-type EEEV.

"A similar phenotype was observed after infection of human K562 and THP-1 monocyte/macrophage cells with wild-type EEEV and [the EEEV mutant], as well as infection of primary bone marrow-derived dendritic cells, reported to express high levels of miR-142-3p," they wrote.

Further experimentation with wild-type and mutant EEEV in the dendritic cells showed that myeloid restriction of wild-type EEEV is independent of interferon levels, but dependent on the 3' NTRs containing miR-142-3p binding sites.

To study how miR-142-3p acts to restrict EEEV replication, the Pitt team expressed the miRNA in BKH-21 cells and examined its effects on infection by wild-type and the mutant EEEV, as well as by VEEV.

"Ectopic expression of miR-142 in BHK-21 cells completely blocked wild-type EEEV infection in comparison to control cells expressing a neuron-specific miRNA, miR-124," according to the paper. On the other hand, both the mutant EEEV and wild-type VEEV infected mirR-142-expression BHK-21 cells.

The researchers next generated mutant viruses with their miR-142-3p binding sequences deleted or with three-point mutations in each miR-142-3p binding site seed sequence. They found that these viruses replicated equally well in both the BHK-21 and RAW cells, pointing to the critical role of the miRNA's binding sites in restricting EEEV replication.

To bear out their findings in vivo, the Pitt investigators turned to EEEV-infected mice, which exhibit minimal early-stage symptoms of infection such as ruffled fur and weight loss. As with human infection, these limited prodromes are likely due to restricted myeloid cell replication and interferon induction.

To determine the role of miR-142-3p to EEEV pathogenesis in rodents, the team infected CD-1 mice with either wild-type EEEV or their mutant strain with the nucleotide deletions, and found that survival times in the mice with the mutant virus were extended compared with the other group.

Because RNA viruses mutate rapidly, the researchers posited that the miR-142-3p binding sites in the EEEV 3' NTR are maintained by positive selection during the mosquito-vertebrate transmission cycle.

In mosquito cells, the EEEV strains with the nucleotide deletions, the binding site deletions, or the three point mutations all showed reduced replication compared with the wild-type virus 12 hours after infection. However, by 24 hours, only the first two mutants remained attenuated.

Furthermore, reduced infection rates of the EEEV bridge vector Ochlerotatus taeniorhynchus through artificial blood meals were observed for all three mutant strains when compared with wild-type EEEV.

As such, "specific sequences of the miR-142-3p binding sites are required for efficient mosquito infection," they determined.

Taken together, the findings point to host miRNA restriction of EEEV replication in myeloid cells as a "novel mechanism that determines virus tropism for this cell lineage," the scientists concluded.

"It is also clear from these data that, at the organism level, miRNA-mediated restriction of virus replication can lead to suppression of innate immune responses and exacerbation of disease, thereby benefiting the infecting microorganism," they concluded.

"Ultimately, these results suggest that the mutant virus could be used as an EEEV vaccine and that microRNA blockers could have potential for use as a therapeutic treatment for EEEV-infected patients who currently can be treated only with supportive care," Pitt's William Klimstra, senior author of the Nature paper, said in a statement.

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